This project is developing an in vivo ?kinase assay?, which is widely applicable to protein kinases. Akt1 and Akt2 is our initial model. The two kinases share high sequence homology, but display functional difference in development, diabetics, cancers, and et al. Our motivation is to determine whether different Akt1 and Akt2 substrate specificity and/or preference are an underlying mechanism. The project will nurture the growth of at least two undergraduate and one graduate students to the next stage of their biomedical career. Briefly, we are combining carbon nanotube delivery of ATP analogues, CRISPR/Cas9 gene editing and chemical-genetics, in order to be able to distinguish the endogenous phosphorylation reactions of the kinase of interest from those of the > 500 kinases encoded in the human genome ? a prerequisite for in vivo ?kinase assay?. We are using a protocol developed by one of us (Dr. Cai) to use carbon nanotubes for intracellular delivery of the ATP analog A*TP-g-S. Akt1 and Akt2 genes are being CRISPR/Cas9 edited to mutate the gatekeeper methionine to a glycine, in order to enlarge their ATP binding pockets. That is, creation of two variants of HCT116 cells anchoring Akt1M?G and Akt2M?G, respectively. According to the well-established chemical-genetic method developed by Dr. Kevan Shokat, only the mutant kinase is able to accommodate the bulky ATP analogue A*TP-g-S due to its modified bulkier adenine moiety. The analogue also has the ATP g-phosphate group replaced by a thiophosphate group. Consequently, nanotube delivered analogue is used exclusively by the mutant kinase to thiophosphate-tag its substrates, both known ones and those that are new and previously unsuspected. The tag can then be used as a handle to isolate the substrates, which can then be identified and quantified with LC-MS/MS in a systematic and unbiased manner. That is, a spectrum of substrates ? the identity and relative abundance of all substrates ? will be identified respectively for Akt1 and Akt2. Comparing the Akt1 and the Akt2 spectrums of substrates in HCT116 cells will reveal whether differential substrate specificities and/or preferences are an underlying mechanism for their functional differences. Our aims to accomplish these research tasks are: 1) to CRISPR/Cas9 edit Akt1 and Akt2 to enlarge their ATP binding pockets in the HCT116 human cells, i.e., creation of Akt1M?G cells and Akt2M?G cells; and 2) to identify and differentiate the spectrums of substrates of the two kinases in HCT116 cells. Significance: The results will set a new and general paradigm for studying protein kinases. Akt1 and Akt2 have long been targeted for drug and therapeutic development. Identification of their respective spectrum of substrates will help guide further development and improvement efforts for relevant human diseases. Additionally, the project provides an excellent interdisciplinary platform for students to integrate classroom education and research activities.